Nitrogenous heterocyclic compounds

ABSTRACT

The present invention relates to nitrogen-containing heterocyclic compounds and pharmaceutically acceptable salts thereof which have inhibitory activity on the phosphorylation of kinases, which inhibits the activity of such kinases. The invention is also related to a method of inhibiting kinases and treating disease states in a mammal by inhibiting the phosphorylation of kinases. In a particular aspect the present invention provides nitrogen-containing heterocyclic compounds and pharmaceutically acceptable salts thereof which inhibit phosphorylation of a PDGF receptor to hinder abnormal cell growth and cell wandering, and a method for preventing or treating cell-proliferative diseases such as arteriosclerosis, vascular reobstruction, cancer and glomerulosclerosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/344,737,filed Mar. 18, 2004, which is a national stage of PCT/US01/41751, filedAug. 17, 2001, which is an application claiming the benefit under 35 USC119(e) of 60/226,089, filed Aug. 18, 2000, the content of which isincorporated herein by reference

TECHNICAL FIELD

The present invention relates to nitrogen-containing heterocycliccompounds and pharmaceutically acceptable salts thereof which haveinhibitory activity on the phosphorylation of kinases, which inhibitsthe activity of such kinases. The invention is also related to a methodof inhibiting kinases and treating disease states in a mammal byinhibiting the phosphorylation of kinases.

BACKGROUND ART

PDGF (platelet-derived growth factor) is known to act as an aggravatingfactor for cell-proliferative diseases such as arteriosclerosis,vascular reobstruction after percutaneous coronary angioplasty andbypass operation, cancer, glomerulonephritis, glomerulosclerosis,psoriasis and articular rheumatism [Cell, 46, 155-169 (1986); Science,253, 1129-1132 (1991); Nippon Rinsho (Japanese J. of Clinical Medicine),50, 3038-3045 (1992); Nephrol Dial Transplant, 10, 787-795 (1995);Kidney International, 43 (Suppl. 39), 86-89 (1993); Journal ofRheumatology, 21, 1507-1511 (1994); Scandinavian Journal of Immunology,27, 285-294 (1988), etc.].

As for quinazoline derivatives which are useful as drugs,N,N-dimethyl-4-(6,7-dimethoxy-4-quinazolinyl)-1-piperazine carboxamideis described as a bronchodilator in South African Patent No. 67 06512(1968). Dimethoxyquinazoline derivatives are described as inhibitors ofphosphorylation of epidermal growth factor (EGF) receptor in JapanesePublished Unexamined Patent Application No. 208911/93 and WO 96/09294.Quinoline derivatives having benzodiazepin receptor agonist activity aredescribed in Pharmacology Biochemistry and Behavior, 53, 87-97 (1996)and European Journal of Medicinal Chemistry, 31, 417-425 (1996), andquinoline derivatives which are useful as anti-parasite agents aredescribed in Indian Journal of Chemistry, 26B, 550-555 (1987).

Inhibitors of phosphorylation of PDGF receptor so far known includebismono- and bicyclic aryl compounds and heteroaryl compounds (WO92/20642), quinoxaline derivatives [Cancer Research, 54, 6106 (1994)],pyrimidine derivatives (Japanese Published Unexamined Patent ApplicationNo. 87834/94) and dimethoxyquinoline derivatives [Abstracts of the 16thAnnual Meeting of the Pharmaceutical Society of Japan (Kanazawa) (1996),2, p. 275, 29(C2) 15-2].

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide nitrogen-containingheterocyclic compounds and pharmaceutically acceptable salts thereofwhich have inhibitory activity on the phosphorylation of kinases, whichinhibits the activity of the kinases. Particularly, important kinaseinhibition according to the invention is of receptor tyrosine kinasesincluding platelet-derived growth factor (PDGF) receptor, Flt3, CSF-1R,epidermal growth factor receptor (EGRF), fibroblast growth factor (FGF),vascular endothelial growth factor receptor (VEGFR) and others. Anotherclass of kinase inhibition according to the invention is inhibitoryactivity nonreceptor tyrosine kinases including src and abl, and thelike. A third class of kinase inhibition according to the invention isinhibitory activity toward serine/threonine kinases, including suchkinases as MAPK, MEK and cyclin dependent kinases (CDKs) that mediatecell prolifetation, AKT and CDK such that mediate cell survival and NIKthat regulate inflammatory responses. Inhibition of such kinases can beused to treat diseases involving cell survival, proliferation andmigration, including cardiovascular disease, such as arteriosclerosisand vascular reobstruction, cancer, glomerulosclerosis fibrotic diseasesand inflammation, as well as the general treatment of cell-proliferativediseases.

In a preferred embodiment, the present invention provides compounds andpharmaceutically acceptable salts thereof which inhibit or preventinhibition of phosphorylation of at least one PDGF receptor by at leastone tyrosine kinase. Such PDGF receptor kinase inhibition can hinderabnormal cell growth and cell wandering, and thus such compounds areuseful for the prevention or treatment of cell-proliferative diseasessuch as arteriosclerosis, vascular reobstruction, cancer andglomerulosclerosis.

The present invention relates to nitrogen-containing heterocycliccompounds represented by formula I as follows:

wherein

-   R¹ is a member selected from the group consisting of:

—CN, —O—C₁₋₈ alkyl that is a straight or branched chain, —O-phenyl,—O-naphthyl, —O-indolyl and —O-isoquinolinyl;

-   R² and R⁴ are each independently a member selected from the group    consisting of:

hydrogen, —O(—CH₂)—CH₃, —O—CH₂—CH═CH₂, —O—CH₂—C≡CH and —O(—CH₂)_(n)—R³;providing that one of the R² and R⁴ groups is hydrogen and the remainingR² or R⁴ group is other than hydrogen;

-   n is 1, 2 or 3;-   R³ is a member selected from the group consisting of:-   —OH, —O—CH₃, —O—CH₂—CH₃, —NH₂, —N(—CH₃)₂, —NH(—CH₂-phenyl),    —NH(-phenyl), —CN

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof.

Particularly preferred compounds according to formula above are suchcompounds wherein R¹ is a member selected from the group consisting ofCN, —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl, —O-t-butyl,—O-isoamyl, 1-naphthyloxy, 2-naphthyloxy, 4-indolyloxy, 5-indolyloxy,5-isoquinolyloxy, and position isomers and homologs thereof, and allpharmaceutically acceptable isomers, salts, hydrates, solvates andprodrug derivatives of such compounds.

The pharmaceutically acceptable salts of the compounds according toformula (I) include pharmaceutically acceptable acid addition salts,metal salts, ammonium salts, organic amine addition salts, amino acidaddition salts, etc. Examples of the pharmaceutically acceptable acidaddition salts of the compounds of formula (I) are inorganic acidaddition salts such as hydrochloride, sulfate and phosphate, and organicacid addition salts such as acetate, maleate, fumarate, tartrate,citrate and methanesulfonate. Examples of the pharmaceuticallyacceptable metal salts are alkali metal salts such as sodium salt andpotassium salt, alkaline earth metal salts such as magnesium salt andcalcium salt, aluminum salt and zinc salt. Examples of thepharmaceutically acceptable ammonium salts are ammonium salt andtetramethyl ammonium salt. Examples of the pharmaceutically acceptableorganic amine addition salts include heterocyclic amine salts such asmorpholine and piperidine salts. Examples of the pharmaceuticallyacceptable amino acid addition salts are salts with lysine, glycine andphenylalanine.

In a preferred embodiment the invention provides compounds according toformula I(a) and formula I(b) as follows:

wherein

-   R¹ is a member selected from the group consisting of:

—CN, —O—C₁₋₈ alkyl that is a straight or branched chain, —O-phenyl,—O-naphthyl, —O-indolyl and —O-isoquinolinyl;

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof.

In another preferred embodiment the invention provides compoundsaccording to formula (Ic) and formula (Id) as follows:

wherein

-   R¹ is a member selected from the group consisting of:

—CN, —O—C₁₋₈ alkyl that is a straight or branched chain, —O-phenyl,—O-naphthyl, —O-indolyl and —O-isoquinolinyl;

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof.

In still another preferred embodiment the invention provides compoundsaccording to formula I(e) and formula I(f) as follows:

wherein

-   R¹ is a member selected from the group consisting of:

—CN, —O—C₁₋₈ alkyl that is a straight or branched chain, —O-phenyl,—O-naphthyl, —O-indolyl and —O-isoquinolinyl;

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof.

In yet another preferred embodiment the invention provides compoundsaccording to formula I(g) and formula I(h) as follows:

wherein

-   R¹ is a member selected from the group consisting of:

—CN, —O—C₁₋₈ alkyl that is a straight or branched chain, —O-phenyl,—O-naphthyl, —O-indolyl and —O-isoquinolinyl;

-   R³ is a member selected from the group consisting of:-   —OH, —O—CH₃, —O—CH₂—CH₃, —NH₂, —N(—CH₃)₂, —NH(—CH₂-phenyl),    —NH(-phenyl), —CN    and all pharmaceutically acceptable isomers, salts, hydrates,    solvates and prodrug derivatives thereof.

The pharmaceutically acceptable salts of the compounds according toformula (I) include pharmaceutically acceptable acid addition salts,metal salts, ammonium salts, organic amine addition salts, amino acidaddition salts, etc.

The present invention is not limited by the above listed compounds.Analogs of the bicyclic compounds are contemplated.

Further, an especially preferred embodiment of the present invention isa compound selected from the group consisting of:

N-[4-(methylethoxy)phenyl]{4-[7-(2-morpholin-4-ylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(2-morpholin-4-ylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(2-pyrrolidinylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(2-pyrrolidinylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-N-[4-(methylethoxy)phenyl]carboxamide

N-(4-cyanophenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carboxamide

{4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-N-(4-phenoxyphenyl)carboxamide

N-(4-indol-5-yloxyphenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-(5-isoquinolyloxy)phenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(3-piperidylpropoxy)quinazolin-4-yl]piperazinyl)carboxamide

{4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}-N-(4-phenoxyphenyl)carboxamide

N-(4-indol-5-yloxyphenyl){4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-(5-isoquinolyloxy)phenyl){4-[7-(2-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(3-piperidylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(3-morpholin-4-ylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(3-morpholin-4-ylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-[4-(methylethoxy)phenyl]{4-[7-(3-pyrrolidinylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(3-pyrrolidinylpropoxy)quinazolin-4-yl]piperazinyl}carboxamide

N-(4-cyanophenyl){4-[7-(3-methoxypropoxy)quinazolin-4-yl]piperazinyl}carboxamide

{4-[7-(3-methoxypropoxy)quinazolin-4-yl]piperazinyl}-N-[4-(methylethoxy)phenyl]carboxamide

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof.

The compounds may be prepared using methods and procedures as generallydescribed in WO 98/14431 published Sep. 12, 1998, which is incorporatedherein by reference. Starting materials may be made or obtained asdescribed therein as well.

Leaving groups such as halogen, lower alkoxy, lower alkylthio, loweralkylsulfonyloxy, arylsulfonyloxy, etc, may be utilized when necessaryexcept for the reaction point, followed by deprotection. Suitable aminoprotective groups are, for example, those described in T. W. Greene,Protective Groups in Organic Synthesis, John Wiley & Sons Inc. (1981),etc., such as ethoxycarbonyl, t-butoxycarbonyl, acetyl and benzyl. Theprotective groups can be introduced and eliminated according toconventional methods used in organic synthetic chemistry [e.g., T. W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons Inc.(1981)].

In such processes, if the defined groups change under the conditions ofthe working method or are not appropriate for carrying out the method,the desired compound can be obtained by using the methods forintroducing and eliminating protective groups which are conventionallyused in organic synthetic chemistry [e.g., T. W. Greene, ProtectiveGroups in Organic Synthesis, John Wiley & Sons Inc. (1981)], etc.Conversion of functional groups contained in the substituents can becarried out by known methods [e.g., R. C. Larock, Comprehensive OrganicTransformations (1989)] in addition to the above-described processes,and some of the active compounds of formula I may be utilized asintermediates for further synthesizing novel derivatives according toformula I.

The intermediates and the desired compounds in the processes describedabove can be isolated and purified by purification methodsconventionally used in organic synthetic chemistry, for example,neutralization, filtration, extraction, washing, drying, concentration,recrystallization, and various kinds of chromatography. Theintermediates may be subjected to the subsequent reaction withoutpurification.

There may be tautomers for some formula I, and the present inventioncovers all possible isomers including tautomers and mixtures thereof.Where chiral carbons lend themselves to two different enantiomers, bothenantiomers are contemplated as well as procedures for separating thetwo enantiomers.

In the case where a salt of a compound of formula I is desired and thecompound is produced in the form of the desired salt, it can besubjected to purification as such. In the case where a compound offormula I is produced in the free state and its salt is desired, thecompound of formula I is dissolved or suspended in a suitable organicsolvent, followed by addition of an acid or a base to form a salt.

The following non-limiting reaction Schemes I, II and III illustratepreferred embodiments of the invention with respect to making compoundsaccording to the invention.

Scheme I describes the synthesis7-(2-methoxyethoxy)-4-piperazinylquinazoline a key intermediate thatwill be utilized in the synthesis of various targets as described inScheme II. The 2-amino-4-fluorobenzoic acid is esterified followed bycyclization with formamide at elevated temperature to afford7-fluoro-4-quinazolinone. The 7-fluoro group is displaced with severalalkoxides generated by treating with NaH in DMF at 100° C. The7-alkoxy-4-quinazolinone is intermediate is converted to7-alkoxy-4-chloroquinazoline with thionyl chloride. The key intermediateI was obtained by treating 7-alkoxysubstituted-4-Cl-quinazoline withpiperazine in an appropriate solvent, such as isopropanol, acetonitrile,or THF at room or reflux temperature for 1-6 h in presence of basetriethylamine or pyridine.

This illustrated Scheme II provides the synthesis of various substitutedurea from the intermediate obtained in Scheme I, or by other procedures.The reaction of intermediate I with various isocyanates afforded thefinal urea compounds. In cases where the isocyanates are notcommercially available, the piperazine intermediate is treated withphosgene to give carbamoyl chloride intermediate followed by reactionwith various substituted anilines. The piperazine intermediate can alsobe treated with p-nitrophenyl chloroformate to afford nitrophenylcarbamate intermediate that can be treated with various anilines toafford the desired ureas.

If the urea compound has a terminal NH₂ group (or one or more of thehydrogen atoms on this amino group is replaced by a displaceablesubstituent), then this compound may be utilized an intermediatecompound with which to produce a urea compound terminated with a—NH-phenyl-R¹ groups. Alternatively, if the a different R¹ group isdesired on the phenyl group, a replaceable para position leaving groupphenyl substituent may be displaced after coupling to provide theparticular R¹ substituent as described for formula I, above.

Scheme III describes the synthesis of6-(2-methoxyethoxy)-4-piperazinylquinazoline as a key intermediate thatwill be utilized in the synthesis of various positional isomer targets.

Such procedures for producing the claimed compounds are merely anillustration of a preferred aspect of the invention. Other proceduresand adaptations will be apparent to one of ordinary skill in the artupon views these reaction schemes and the structures of the compoundsaccording to the invention. Such procedures are deemed to be within thescope of the present invention.

Also, the compounds of formula I and pharmaceutically acceptable saltsthereof may exist in the form of adducts with water (hydrates) orvarious solvents, which are also within the scope of the presentinvention.

The following non-limiting examples are provided to better illustratethe present invention.

EXAMPLE 1

The intermediate 4-piperazinyl-7-(2-piperidylethoxy)quinazoline wasprepared using the procedures as generally described in Scheme 1 asfollows:

Step A: To the ethanol solution (15 mL) of 2-amino-4-fluorobenzoic acid(840 mg, 5.41 mmmol) was added thionyl chloride (1.18 mL, 16.23 mmol)and the resulting suspension was refluxed overnight. The solvent wasevaporated to the residue added EtOAc, washed with 10% NaOH solution,dried, filtered and evaporated to afford desired ethyl ester as a solid981 mg, 82%). MS (ES) 184(M+H)

Step B: To the formamide (6 mL) solution ofethyl-2-amino-4-fluorobenzoate (811 mg, 4.43 mmol) added ammoniumformate (0.450 g, 7.14 mmol) and the reaction mixture was heated at 140°C. overnight. After cooling added water and ethyl acetate. The layerswere separated, the EtOAc layer was dried, filtered and evaporated togive desired quinazolinone (1 g, quantitative). MS (ES) 165 (M+H)

Step C: To the DMF solution (3 mL) of 1-piperidineethanol (0.689 mL,5.18 mmol) at 0° C. added sodium hydride (0.518 g, 12.95 mmol) and themixture was stirred for 30 min. To this cold solution added DMF solution(3 mL) of intermediate quinazolinone (0.284 g, 1.73 mmol, from Step B)and the mixture was heated at 75° C. overnight. The solvent wasevaporated and the residue purified by RP-HPLC to afford desired productas a creamy solid (0.459 g, 97%)

Step D: A mixture of 7-(2-piperidylethoxy)-4-quinazolinone (0.459 g,1.68 mmol), from Step C) and POCl₃ (5 mL) was heated at 75° C.overnight. After cooling excess POCl₃ was removed by evaporation and theresidue azeotroped with toluene to afford intermediate,4-chloro-7-(2-piperidylethoxy)quinazoline (600 mg, 95%)

Step E: To the isopropanol solution (10 mL) of 4-chloroquinazoline(0.615 g, 2.11 mmol, from Step D) added piperazine (0.727 g, 8.44 mmol)and heated the reaction for 4 h at 100° C. The solvent was evaporatedand the residue purified by RP-HPLC to afford4-piperazinyl-7-(2-piperidylethoxy)quinazoline as a white solid (0.630g, 87%).

EXAMPLE 2

Preparation of N-(4-cyanophenyl){4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}-carboxamide

To DMF solution (2 mL) of 4-piperazinyl-7-(2-piperidylethoxy)quinazoline(from Example 1, Step E, 0.287 g, 0.84 mmol) added DMF solution (2 mL)of 4-cyanophenylisocyanate (0.181 g, 1.26 mmol) and the reaction wasstirred at room temperature overnight. The solvent was evaporated andresidue purified by RP-HPLC to afford desired product N-(4-cyanophenyl){4-[7-(2-piperidylethoxy)quinazolin-4-yl]piperazinyl}carboxamide as awhite solid (200 mg, 50%). MS (ES) 487(M+H)

EXAMPLES 3-4

N-(4-cyanophenyl){4-[7-(2-methoxyethoxy)quinazolin-4-yl]piperazinyl}-carboxamide wasprepared using the procedures as described above in Example 1 and 2except that 2-methoxyethanol was used instead of 1-piperidineethanol asan alkoxide anion, to provide the title compound.

The pharmacological activities of the compounds of the present inventionare obtained by following the test example procedures as follows, forexample.

Biological Test Assay Type 1

Inhibitory Effect on Compounds on Autophosphorylation of PlateletDerived Growth Factor β-PDGF Receptor

(1) HR5 Phosphorylation Assay

The HR5 cell line is a cell line of CHO cells engineered to overexpresshuman β-PDGFR, which cell line is available from the ATCC. Theexpression level of β-PDGFR in HR5 cells is around 5×10⁴ receptor percell. For the phosphorylation assay according to the invention, HR5cells were grown to confluency in 96-well microtiter plates understandard tissue culture conditions, followed by serum-starvation for 16hours. Quiescent cells were incubated at 37° C. without or withincreasing concentrations of the test compound (0.01-30 uM) for 30minutes followed by the addition of 8 nM PDGF BB for 10 minutes. Cellswere lysed in 100 mM Tris, pH7.5, 750 mM NaCl, 0.5% Triton X-100, 10 mMsodium pyrophosphate, 50 mM NaF, 10 ug/ml aprotinin, 10 ug/ml leupeptin,1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and the lysatewas cleared by centrifugation at 15,000×g for 5 minutes. Clarifiedlysates were transferred into a second microtiter plate in which thewells were previously coated with 500 ng/well of 1B5B11 anti-β-PDGFRmAb, and then incubated for two hours at room temperature. After washingthree times with binding buffer (0.3% gelatin, 25 mM Hepes pH 7.5, 100mM NaCl, 0.01% Tween-20), 250 ng/ml of rabbit polyclonalanti-phosphotyrosine antibody (Transduction Laboratory) was added andplates were incubated at 37° C. for 60 minutes. Subsequently, each wellwas washed three times with binding buffer and incubated with 1 ug/ml ofhorse radish peroxidase-conjugated anti-rabbit antibody (BoehringerMannheim) at 37° C. for 60 minutes. Wells were washed prior to addingABTS (Sigma), and the rate of substrate formation was monitored at 650nm. The assay results are reported as IC₅₀ (expressed as theconcentration of a compound according to the invention that inhibits thePDGF receptor phosphorylation by 50%) as compared to control cells thatare not exposed to a compound according to the invention.

Examples of such IC₅₀ test results in the HR5 assay for compoundsaccording to the invention are set forth below in Table 1.

(2) MG63 Phosphorylation Assay

The MG63 cell line is a human osteosarcoma tumor cell line availablefrom the ATCC. This assay is for measuring endogenous β-PDGFRphosphorylation in MG63 cells. The assay conditions are the same asthose described at for HR5 cell, except that PDGF-BB stimulation isprovided in the presence or absence of 45% human plasma. The HR5 assayresults are reported as an IC₅₀ (expressed as the concentration of acompound according to the invention that inhibits the PDGF receptorphosphorylation by 50%) as compared to control cells that are notexposed to a compound according to the invention.

Examples of such IC₅₀ test results in the MG63 assay for compoundsaccording to the invention are set forth below in Table 1.

The assay results for Compound Examples 1 and 2 are set forth in Table 1below. TABLE 1 MG63 w/human plasma HR5 Example Compound IC₅₀ (μM) IC₅₀(μM) Example 1 0.103 0.150 Example 2 3.56 2.27Biological Test Assay Type 2Growth Inhibition Against Smooth Muscle Cells

Vascular smooth muscle cells are isolated from a pig aorta byexplanation and used for the test. The cells are put into wells of a96-well plate (8000 cells/well) and cultured in Dulbeccois modifiedEagle's medium (DMEM; Nissui Pharmaceutical Co., Ltd.) containing 10%fetal bovine serum (FBS; Hyclone) for 4 days. Then, the cells arefurther cultured in DMEM containing 0.1% FBS for 3 days, and aresynchronized at the cell growth stationary phase.

To each well is added DMEM containing 0.1% FBS and a test sample at avaried concentration, and the cell growth is brought about by PDGF-BB(SIGMA, final concentration: 20 ng/ml). After culturing for 3 days, thecell growth is measured using a cell growth assay kit (BoehringerMannheim) according to the XTT method [J. Immunol. Methods, 142, 257-265(1991)], and the cell growth score is calculated by the followingequation.Cell growth score=100×{1−(M−PO)/(P100−PO)}wherein P100=absorbance by XTTreagent when stimulated by PDGF-BB; PO=absorbance by XTT reagent whennot stimulated by PDGF-BB, and M=absorbance by XTT reagent afteraddition of a sample when stimulated by PDGF-BB.

The test result is expressed as the concentration of a test compoundwhich inhibits the cell growth by 50% (IC₅₀).

Biological Test Assay Type 3

Inhibitory Effect on Hypertrophy of Vascular Intima

Male SD rats (weight: 375-445 g, Charles River, golden standard) areanesthetized with sodium pentobarbital (50 mg/kg, i.p.), and then theneck of each animal is incised by the median incision, followed byretrograde insertion of a balloon catheter (2F, Edwards Laboratories)into the left external carotid. After the above treatment is repeatedseven times, the catheter is pulled out, the left external carotid isligated, and the wound is sutured. A test compound is suspended in a0.5% solution of Tween 80 in an aqueous solution of sodium chloride to aconcentration of 20 mg/ml in the case of intraperitoneal administrationand in a 0.5% solution of methyl cellulose 400 to a concentration of 6mg/ml in the case of oral administration. The suspension is administeredonce a day in the case of intraperitoneal administration and once ortwice a day in the case of oral administration for a period of 15 daysstarting on the day before the balloon injury. On the 14th day after theballoon injury, the animal is killed and its left carotid is extirpated.The tissues are fixed with formalin, wrapped in paraffin and sliced,followed by Elastica Wangeeson staining. The area of the cross sectionof the vascular tissues (intima and media) is measured with an imageanalyzer (Luzex F, NIRECO) and the intima/media area ratio (I/M) isregarded as the degree of hypertrophy of the vascular intima.

From the results obtained, it is apparent when the hypertrophy ofvascular intima is significantly inhibited by administration of thecompounds of the present invention.

Biological Test Assay Type 4

Evaluation by the Use of a Rat Adjuvant Arthritis Model

Dead cells of Mycobacterium bacterium (Difco Laboratories Inc.) aredisrupted in agate mortar and suspended in liquid paraffin to the finalconcentration of 6.6 mg/ml, followed by sterilization with high pressuresteam. Then, 100 ml of the suspension is subcutaneously injected intothe right hind foot pad of each animal of groups of female 8-weeks-oldLewis rats (Charles River Japan) (6 animals/group) to induce adjuvantarthritis. A test compound is suspended in a 0.5% solution of methylcellulose to the final concentration of 3 mg/ml, and from just beforethe induction of arthritis, the suspension is orally administered in anamount of 100 ml/100 g of the body weight once a day, 5 days a week. Toa control group is administered a 0.5% solution of methyl cellulose. Anormal group is given no adjuvant treatment or test compoundadministration. The administration of the test compound is continuedtill the 18th day after the adjuvant treatment. On the 17th day, thenumber of leukocytes in peripheral blood are counted, and on the 18thday, all the blood is collected, followed by dissection.

The change in body weight with the passage of time, the change of edemain hind foot with the passage of time, the weight of spleen and thymus,the number of leukocytes in peripheral blood, the hydroxyproline contentof urine, the glucosaminoglycan content of urine, the SH concentrationin serum, the concentration of nitrogen monoxide in serum and theconcentration of mucoprotein in serum are measured and evaluated. Thevolume of each of both hind feet are measured using a rat's hind footedema measurement device (TK-101, Unicom). The number of leukocytes inperipheral blood are counted using an automatic multichannel blood cellcounter (Sysmex K-2000, Toa Iyo Denshi Co., Ltd.). The hydroxyprolinecontent of urine is measured according to the method described in Ikeda,et al., Annual Report of Tokyo Metropolitan Research Laboratories P. H.,, 36, 277 (1985), and the glucosaminoglycan content is measuredaccording to the method described in Moriyama, et al., Hinyo Kiyo, 40,565 (1994) and Klompmakers, et al., Analytical Biochemistry, 153, 80(1986). The SH concentration in serum is measured according to themethod described in Miesel, et al., Inflammation, 17, 595 (1993), andthe concentration of nitrogen monoxide is measured according to themethod of Tracey, et al., Journal of Pharmacology & ExperimentalTherapeutics, 272, 1011 (1995). The concentration of mucoprotein ismeasured using Aspro GP Kit (Otsuka Pharmaceutical Co., Ltd.). Thepercentage inhibition for each indication is calculated according to thefollowing equation.% Inhibition={(Control group−Compound-administered group)/(Controlgroup−Normal group)}×100.

From the results obtain from such assays, it is apparent when thecompound according to the invention inhibits the occurrence of adjuvantarthritis.

Biological Test Assay Type 5

Activity on a Mesangial Proliferative Glomerulonephritis Model

Anti-rat Thy-1.1 monoclonal antibody OX-7 (Sedaren) is administered tomale Wister-Kyoto rats (Charles River Japan, 160 g, 6 animals/group) inan amount of 1.0 mg/kg by intravenous administration through the tailvein. A test compound is suspended in a 0.5% solution of methylcelluloseand the resulting suspension is administered to each of the rats twice aday for a period of 7 days starting on the day before the administrationof OX-7. On the 7th day after the OX-7 administration, when mesangialcell growth and extracellular matrix hypertrophy become prominent, theleft kidney of each rat is extirpated, fixed with 20% buffered formalinfor 6 hours and wrapped in paraffin, followed by slicing. The obtainedpieces are subjected to immune tissue staining using antibody PC10(DAKO) against an intranuclear antigen of proliferative cells. Aftercomparative staining with Methyl Green staining solution usingdiaminobenzidine as a color developer, the paraffin pieces are enclosed.Half of the glomeruli in a kidney piece are observed and the number ofthe cells in one glomerulus which are positive to the intranuclearantigen of proliferative cells are calculated. The test for thesignificance of difference is carried out by the Wilcoxon test.

From such results, it is apparent when the compounds according to thepresent invention show alleviating activity on mesangial proliferativeglomerulonephritis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof can be administered as such, but it is usually preferred toadminister them in the form of pharmaceutical compositions, which areused for animals and human beings.

It is preferred to employ the administration route which is the mosteffective for the treatment. For example, administration is made orallyor non-orally by intrarectal, intraoral, subcutaneous, intramuscular orintravenous administration.

Examples of the forms for administration are capsules, tablets,granules, powders, syrups, emulsions, suppositories and injections.

Liquid compositions such as emulsions and syrups which are appropriatefor oral administration can be prepared using water, sugars such assucrose, sorbitol and fructose, glycols such as polyethylene glycol andpropylene glycol, oils such as sesame oil, olive oil and soybean oil,preservatives such as benzoates, flavors such as strawberry flavor andpeppermint, etc.

Capsules, tablets, powders and granules can be prepared using excipientssuch as lactose, glucose, sucrose and mannitol, disintegrating agentssuch as starch and sodium alginate, lubricants such as magnesiumstearate and talc, binders such as polyvinyl alcohol, hydroxypropylcellulose and gelatin, surfactants such as fatty acid esters,plasticizers such as glycerin, etc.

Compositions suitable for non-oral administration preferably comprise asterilized aqueous preparation containing an active compound which isisotonic to the recipient's blood. For example, injections are preparedusing a carrier which comprises a salt solution, a glucose solution, ora mixture of a salt solution and a glucose solution.

Compositions for topical application are prepared by dissolving orsuspending an active compound in one or more kinds of solvents such asmineral oil, petroleum and polyhydric alcohol, or other bases used fortopical drugs.

Compositions for intestinal administration are prepared using ordinarycarriers such as cacao fat, hydrogenated fat and hydrogenated fatcarboxylic acid, and are provided as suppositories.

The compositions for non-oral administration may additionally beformulated to contain one or more kinds of additives selected fromglycols, oils, flavors, preservatives (including antioxidants),excipients, disintegrating agents, lubricants, binders, surfactants andplasticizers which are used for the preparation of compositions for oraladministration.

The effective dose and the administration schedule for each of thecompounds of formula (I) or a pharmaceutically acceptable salt thereofwill vary depending on the administration route, the patient's age andbody weight, and the type or degree of the diseases to be treated.However, it is generally appropriate to administer a compound of formula(I) or a pharmaceutically acceptable salt thereof in a dose of 0.01-1000mg/adult/day, preferably 5-500 mg/adult/day, in one to several parts.

All the compounds of the present invention can be immediately applied tothe treatment of kinase-dependent diseases of mammals as kinaseinhibitors, specifically, those relating to tyrosine kinase.Specifically preferred are the compounds which have IC50 within therange of 10 nM-10 μM. Even more preferred are compounds which have IC50within the range of 10 μM to −1 μM. Most preferred are compounds whichhave an IC50 value which is smaller than 1 μM.

Specific compounds of the present invention which have an activity tospecifically inhibit one of the three types of protein kinase (forexample, kinase which phosphorylates tyrosine, kinase whichphosphorylates tyrosine and threonine, and kinase which phosphorylatesthreonine) can be selected. Tyrosine kinase-dependent diseases includehyperproliferative malfunction which is caused or maintained by abnormaltyrosine kinase activity. Examples thereof include psoriasis, pulmonaryfibrosis, glomerulonephritis, cancer, atherosclerosis andanti-angiopoiesis (for example, tumor growth and diabetic retinopathy).Current knowledge of the relationship between other classes of kinaseand specific diseases is insufficient. However, compounds havingspecific PTK-inhibiting activity have a useful treatment effect. Otherclasses of kinase have also been recognized in the same manner.Quercetin, genistein and staurosporin, which are all PTK-inhibitors,inhibit many kinds of protein kinase in addition to tyrosine kinase.However, as a result of their lack of the specificity, theircytotoxicity is high. Therefore, a PTK-inhibitor (or an inhibitor ofother classes of kinase) which is apt to bring about undesirable sideeffects because of the lack of selectivity can be identified by the useof an ordinary test to measure cytotoxicity.

The present invention provides nitrogen-containing heterocycliccompounds and pharmaceutically acceptable salts thereof which inhibitphosphorylation of PDGF receptor to hinder abnormal cell growth and cellwandering and thus are useful for the prevention or treatment ofcell-proliferative diseases such as arteriosclerosis, vascularreobstruction, cancer and glomerulosclerosis.

Although the present invention has been described in some detail by wayof illustration for purposes of clarity of understanding, it will beapparent to those of ordinary skill in the art that variousmodifications and equivalents can be made without departing from thespirit and scope of the invention. It should be understood that theforegoing discussion and examples merely present a detailed descriptionof certain preferred embodiments. All the patents, journal articles andother documents discussed or cited above are herein incorporated byreference in their entirety.

1. A method of inhibiting phosphorylation of PDGF receptor in a patientcomprising the step of administering a compound selected from the groupconsisting of:

wherein when the compound is of Formula I(a) or I(b), R¹ is a memberselected from the group consisting of: CN, O-methyl, —O-ethyl,—O-propyl, —O-isopropyl, —O-butyl, —O-t-butyl, —O-isoamyl,1-naphthyloxy, 2-naphthyloxy, 4-indolyloxy, 5-indolyloxy,5-isoquinolyloxy, and position isomers and homologs thereof; and whenthe compound is of Formula I(c), I(d), I(e), I(f), I(g) or I(h), R¹ is amember selected from the group consisting of: CN, —O—C₁₋₈alkyl that isstraight or branched chained, —O-phenyl, -naphthyloxy, -indolyloxy and-isoquinolinyloxy; n is 1, 2 or 3; R³ is a member selected from thegroup consisting of: —OH, —O—CH₃, —O—CH₂—CH₃, —NH₂, —N(—CH₃)₂,—NH(—CH₂-phenyl), —NH(-Phenyl), —CN

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof to the patient.
 2. A method forinhibiting abnormal cell growth and cell wandering in a patient andthereby preventing or treating a cell-proliferative disease, comprisingthe step of administering a compound selected from the group consistingof:

wherein when the compound is of Formula I(a) or I(b), R¹ is a memberselected from the group consisting of: CN, O-methyl, —O-ethyl,—O-propyl, —O-isopropyl, —O-butyl, —O-t-butyl, —O-isoamyl,1-naphthyloxy, 2-naphthyloxy, 4-indolyloxy, 5-indolyloxy,5-isoquinolyloxy, and position isomers and homo logs thereof; and whenthe compound is of Formula I(c), I(d), I(e), I(f), I(g) or I(h), R¹ is amember selected from the group consisting of: CN, —O—C₁₋₈alkyl that isstraight or branched chained, —O-phenyl, -naphthyloxy, -indolyloxy and-isoquinolinyloxy; n is 1, 2 or 3; R³ is a member selected from thegroup consisting of: —OH, —O—CH₃, —O—CH₂—CH₃, —NH₂, —N(—CH₃)₂,—NH(—CH₂-phenyl), —NH(-phenyl), —CN

and all pharmaceutically acceptable isomers, salts, hydrates, solvatesand prodrug derivatives thereof to the patient.
 3. A method according toclaim 2, wherein said cell-proliferative disease is selected from thegroup consisting of arteriosclerosis, vascular re-obstruction,restenosis, cancer and glomerulosclerosis.